Reagent reservoir system for analytical instruments

ABSTRACT

The invention provides a reagent reservoir system and disposable reaction cassettes using the same. In one aspect, such system comprises a chamber in which dried reagent, particularly lyophilized reagent, is constrained to remain in a defined region of the chamber by a retaining member that obstructs passage of such reagents to other regions of the chamber where they may escape hydration or activation.

This application is a division of U.S. patent application Ser. No.13/458, 762, filed Apr. 27, 2012, U.S. Pat. No. 8,758,701 which is adivision of U.S. patent application Ser. No. 11/485,944, filed Jul. 13,2006, now U.S. Pat. No. 8,187,557, the content of such application isincorporated herein by reference in its entirety.

FIELD OF THE INVENTION

The invention relates generally to methods and apparatus for storing andactivating reagents for analytical assays, and more particularly, tomethods and apparatus for storing and activating dried or lyophilizedreagents.

BACKGROUND

Many important assays involve the use of labile reagents, such aspeptides, enzymes, antibodies, or other compounds that are readilydegraded outside narrow environmental ranges. Such assays includeenzyme-based nucleic acid amplification assays, immunoassays, enzymesubstrate assays, and the like. The sensitivity of these reagents notonly makes routine handling and transport more difficult, but also oftenlimits the application of such assays outside of research settings,since opportunities are not available to provide fresh reagents or tomonitor the properties or activities of the reagents. These aresignificant limitations in view of the great interest in using theseassays for a host of important monitoring tasks, frequently in outdoorsettings, where transport, environmental control, and ready access toinstrumentation used with the assays may be limited or unavailable.Examples of such monitoring applications include bio-defense monitoring,agricultural and livestock monitoring, pathogen testing, acute caremedical applications, and the like.

Some problems related to the lability of protein assay reagents havebeen addressed by lyophilization, or freeze-drying, e.g. Franks et al,U.S. Pat. No. 5,098,893; Cole, U.S. Pat. No. 5,102,788; Shen et al, U.S.Pat. No. 5,556,771; Treml et al, U.S. Pat. No. 5,763,157; De Rosier etal, U.S. Pat. No. 6,294,365; Buhl et al, U.S. Pat. No. 5,413,732; andthe like. However, when freeze-dried reagents are used as powders orother small particulates, difficulties can arise in controlling theirdisposition in containers or reaction chambers because of static chargescarried by the particulates, e.g. Matsusaka et al (2002), AdvancedPowder Technol., 13: 157-166. This is particularly troublesome indisposable plastic or solid polymer cartridges that are pre-loaded withsuch particulates or powders and can easily lead tocartridge-to-cartridge measurement variability because the lyophilizedparticulates become inappropriately dispersed and are notrehydratedcompletely.

It would be useful if reagent storage components or cartridges wereavailable that could be pre-loaded with lyophilized particulates, orlike materials, and that would permit complete re-hydration of reagentsin desired concentrations and amounts with minimum possibility of beingdispersed to areas out of contact with a re-hydrating or activatingsolution.

SUMMARY OF THE INVENTION

In one aspect, the invention provides a reagent reservoir system inwhich dried reagents are constrained to remain in a defined region of achamber by a simple inert member that obstructs passage of such reagentsto regions where they may escape hydration or activation. In one aspect,the invention includes a reagent reservoir system comprising: (i) achamber for holding a reagent, the chamber having a longitudinal axis, afluid port, a reagent holding end, and an exhaust port that permits gasin the chamber to exit when a liquid is transferred to the chamberthrough the fluid port, the exhaust port being disposed in the chamberat an end opposite to that of the reagent holding end with respect tothe longitudinal axis; (ii) a dried reagent disposed in the reagentholding end of the chamber, the dried reagent being capable ofdissolving in liquid transferred to the chamber; and (iii) a retainingmember movably disposed in the chamber between the exhaust port and thedried reagent, the retaining member obstructing passage of the driedreagent so that when activation liquid is transferred to the chamber andcollects in the reagent holding end of the chamber the dried reagentdissolves in the liquid to activate the reagent without passing theretaining member.

In another aspect, the invention includes a disposable reaction cassettefor conducting an assay on a sample, such disposable reaction cassettecomprising: (i) a lysis reservoir having a fluid port, the lysisreservoir being capable of holding a lysis buffer; (ii) a sample chamberhaving a sealable port at a first end and a fluid port at a second end,the sample chamber being capable of forming a fluidly closed chamberwhenever the sealable port is closed and the sample chamber beingselectably in fluid communication with the lysis reservoir; (iii) areagent chamber having an exhaust port at a first end, a fluid port anda dried reagent at a second end, and movably disposed therebetween, aretaining member that obstructs passage of the dried reagent through thechamber, wherein the reagent chamber is selectably in fluidcommunication with the sample chamber; and (iv) a reaction chamberhaving an inlet and an outlet, the reaction chamber being selectably influid communication with the reagent chamber.

In still another aspect, the invention provides a reagent reservoir forstoring and activating a dried reagent, the reagent reservoircomprising: (i) a chamber having a first end and a second end along alongitudinal axis, the second end being capable of holding driedreagent; (ii) an exhaust port at the first end; (iii) a fluid port atthe second end; and (iv) a retaining member movably disposed between thefirst and second ends, such that when dried reagent is held in thesecond end, the retaining member obstructs passage of the dried reagentto the first end. In a preferred embodiment, the retaining member isbuoyant in liquid (e.g., has a density less than the density of waterwhich is 1,000 kg/m³) such that when an activation liquid is transferredto the second end by the fluid port, the buoyant retaining member isbuoyed to a top surface of the liquid.

In another aspect, dried reagents for use with the invention includelyophilized particulates, especially those containing reagents forenzyme-based assays, such as nucleic acid amplification assays.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A-1C illustrate several embodiments and the operation of areagent reservoir system of the invention.

FIGS. 2A-2B diagrammatically illustrate an embodiment of a disposablereaction cassette that employs a reagent reservoir system of theinvention.

DEFINITIONS

Terms and symbols of nucleic acid chemistry, biochemistry, genetics, andmolecular biology used herein follow those of standard treatises andtexts in the field, e.g. Kornberg and Baker, DNA Replication, SecondEdition (W.H. Freeman, New York, 1992); Lehninger, Biochemistry, SecondEdition (Worth Publishers, New York, 1975); Strachan and Read, HumanMolecular Genetics, Second Edition (Wiley-Liss, New York, 1999);Eckstein, editor, Oligonucleotides and Analogs: A Practical Approach(Oxford University Press, New York, 1991); Gait, editor, OligonucleotideSynthesis: A Practical Approach (IRL Press, Oxford, 1984); Sambrook etal, Molecular Cloning: A Laboratory Manual, 2nd Edition (Cold SpringHarbor Laboratory, 1989); and the like.

“Amplicon” means the product of a polynucleotide amplification reaction.That is, it is a population of polynucleotides, usually double stranded,that are replicated from one or more starting sequences. The one or morestarting sequences may be one or more copies of the same sequence, or itmay be a mixture of different sequences. Amplicons may be produced by avariety of amplification reactions whose products are multiplereplicates of one or more target nucleic acids. Generally, amplificationreactions producing amplicons are “template-driven” in that base pairingof reactants, either nucleotides or oligonucleotides, to a targetsequence or its complement is required for the creation of reactionproducts. In one aspect, template-driven reactions are primer extensionswith a nucleic acid polymerase or oligonucleotide ligations with anucleic acid ligase. Such reactions include, but are not limited to,polymerase chain reactions (PCRs), linear polymerase reactions, ligasechain reactions (LCRs), strand-displacement reactions (SDAs), nucleicacid sequence-based amplification (NASBAs), rolling circleamplifications, and the like, disclosed in the following references thatare incorporated herein by reference: Mullis et al, U.S. Pat. Nos.4,683,195; 15 4,965,188; 4,683,202; 4,800,159 (PCR); Gelfand et al, U.S.Pat. No. 5,210,015 (real-time PCR with “taqman” probes); Wittwer et al,U.S. Pat. No. 6,174,670; Landegren et al, U.S. Pat. No. 4,988,617(“LCR”); Birkenmeyer et al, U.S. Pat. No. 5,427,930 (“gap-LCR”); Kacianet al, U.S. Pat. No. 5,399,491 (“NASBA”); Walker, U.S. Pat. Nos.5,648,211; 5,712,124 (“SDA”); Lizardi, U.S. Pat. No. 5,854,033; Aono etal, Japanese patent publ. JP 4-262799 (rolling circle 20 amplification);and the like. In one aspect, amplicons are produced in atemperature-cycling amplification reaction that includes repeated stepsof denaturing reaction products, usually double stranded DNA, at a firsttemperature, and annealing primers for polymerase extension at a secondtemperature. A temperature-cycling amplification reactions of specialinterest are PCRs. An amplification reaction may be a “real-time”amplification if a detection chemistry is available that permits areaction product to be measured as the amplification reactionprogresses, e.g. “real-time PCR” described below, or “real-time NASBA”as described in Leone et al, Nucleic Acids Research, 26: 2150-2155(1998), and like references. As used herein, the term “amplifying” meansperforming an amplification reaction. A “reaction mixture” means asolution containing all the necessary reactants for performing areaction, which may include, but not be limited to, buffering agents tomaintain pH at a selected level during a reaction, salts, co-factors,scavengers, and the like.

“Closed” in reference to an amplification reaction means that suchreaction takes place within a vessel or container or chamber that has noopenings through which liquids may pass, in particular, liquids thatcontain non-sample materials, such as, non-sample biomolecules ororganisms, including, but not limited to, nucleic acids, proteins,viruses, bacteria, or the like. In one aspect, a vessel, chamber, orcontainer containing a closed amplification reaction may include a portor vent that is gas permeable but liquid impermeable, for example, aport that permits the venting of air through a filter membrane but notliquids under conventional reaction conditions. Suitable membranes forsuch ports or vents include woven polyolefin films, such as Tyrek® film(DuPont), or the like.

“Fluidly closed” means that, under conventional operating conditions,liquids within a system that comprises one or more vessels, chambers,valves, and/or passages, possibly interconnected and in communicationwith one another, cannot communicate with the exterior of such a system,and likewise liquids on the exterior of such a system cannot communicatewith liquids contained within the interior of the system. In one aspect,conventional operating conditions means that vessels, chambers, valves,and passages of a fluidly closed system are pressurized to an extentless than 1 00 psi, or in another aspect, to an extent less than 50 psi,or to an extent less than 30 psi.

“Indicator” means a probe that is capable of generating an opticalsignal in the presence of a product of an amplification reaction (i.e.an “amplification product”) such that as product accumulates in thereaction mixture the optical signal of the indicator increases, at leastover a predetermined range of concentrations. The optical signalsincludes, but are not limited to, fluorescent signals, chemiluminescentsignals, electrochernilurninescent signals, colorimetric signals, andthe like. “Fluorescent indicator” means an indicator capable ofgenerating a fluorescent signal in the presence of a product of anamplification reaction (i.e. an “amplification product”) such that asproduct accumulates in the reaction mixture the signal of thefluorescent indicator increases, at least over a predetermined range ofconcentrations. Fluorescent indicators may be non-specific, such asintercalating dyes that bind to double stranded DNA products, e.g.YO-PRO-I, SYBR green 1, and the like, Ishiguro et al, Anal. Biochem.,229: 207-213 (1995); Tseng et al, Anal. Biochem., 245: 207-212 (1997);Morrison et al, Biotechniques, 24: 954-962 (1998); or such as primershaving hairpin structures with a fluorescent molecule held in proximityto a fluorescent quencher until forced apart by primer extension, e.g.Whitecombe et al, Nature Biotechnology, 17: 804-807 30(1999)(“Amplifluor™ primers”). Fluorescent indicators also may be targetsequence specific, usually comprising a fluorescent molecule inproximity to a fluorescent quencher until an oligonucleotide moiety towhich they are attached specifically binds to an amplification product,e.g. Gelfand et al, U.S. Pat. No. 5,210,015 (“taqman”); Nazarenko et al,Nucleic Acids Research, 25: 2516-2$21 (1997)(“scorpion probes”); Tyagiet al, Nature Biotechnology, 16: 49-53 (1998)(“molecular beacons”).Fluorescent indicators may be used in connection with real-time PCR, orthey may be used to measure the total amount of reaction product at thecompletion of a reaction.

“Internal standard” means a nucleic acid sequence that is amplified inthe same amplification reaction as a target polynucleotide in order topermit absolute or relative quantification of the target polynucleotidein a sample. An internal standard may be endogenous or exogenous. Thatis, an internal standard may occur naturally in the sample, or it may beadded to the sample prior to amplification. In one aspect, multipleexogenous internal standard sequences may be added to a reaction mixturein a series of predetermined concentrations to provide a calibration towhich a target amplicon may be compared to determine the quantity of itscorresponding target polynucleotide in a sample. Selection of thenumber, sequences, lengths, and other characteristics of exogenousinternal standards is a routine design choice for one of ordinary skillin the art. Preferably, endogenous internal standards, also referred toherein as “reference sequences,” are sequences natural to a sample thatcorrespond to minimally regulated genes that exhibit a constant and cellcycle-independent level of transcription, e.g. Selvey et al, Mol. CellProbes, 15: 307-311 (2001). Exemplary reference sequences include, butare not limited to, sequences from the following genes: GAPDH, beta2-microglobulin, 18S ribosomal RNA, and beta-actin (although see Selveyet al, cited above).

“Kit” refers to any delivery system for delivering materials or reagentsfor carrying out a method of the invention. In the context of reactionassays, such delivery systems include systems that allow for thestorage, transport, or delivery of reaction reagents (e.g., probes,enzymes, etc. in the appropriate containers) and/or supporting materials(e.g., buffers, written instructions for performing the assay etc.) fromone location to another. For example, kits include one or moreenclosures (e.g., boxes) containing the relevant reaction reagentsand/or supporting materials. Such contents may be delivered to theintended recipient together or separately. For example, a firstcontainer may contain an enzyme for use in an assay, while a secondcontainer contains probes. In the context of the present invention, kitsinclude a disposable reaction cassette comprising a reagent reservoirsystem of the invention.

“Ligation” means to form a covalent bond or linkage between the terminiof two or more nucleic acids, e.g. oligonucleotides and/orpolynucleotides, in a template-driven reaction. The nature of the bondor linkage may vary widely and the ligation may be carried outenzymatically or chemically. As used herein, ligations are usuallycarried out enzymatically to form a phosphodiester linkage between a 5′carbon of a terminal nucleotide of one oligonucleotide with 3′ carbon ofanother oligonucleotide. A variety of template-driven ligation reactionsare described in the following references, which are incorporated byreference: Whitely et al, U.S. Pat. No. 4,883,750; Letsinger et al, U.S.Pat. No. 5,476,930; Fung et al, U.S. Pat. No. 5,593,826; Kool, U.S. Pat.No. 5,426,180; Landegren et al, U.S. Pat. No. 5,871,921; Xu and Kool,Nucleic Acids Research, 27: 875-881 (1999); Higgins et al, Methods inEnzymology, 68: 50-71 (1979); Engler et al, The Enzymes, 15: 3-29(1982); and Namsaraev, U.S. patent publication 2004/0110213.

“Microfluidics device” means an integrated system of one or morechambers, ports, and channels that are interconnected and in fluidcommunication and designed for carrying out an analytical reaction orprocess, either alone or in cooperation with an appliance or instrumentthat provides support functions, such as sample introduction, fluidand/or reagent driving means, temperature control, and a detectionsystem. Microfluidics may further include valves, pumps, and specializedfunctional coatings on their interior walls, e.g. to prevent adsorptionof sample components or reactants, facilitate reagent movement byelectroosmosis, or the like. Such devices are usually fabricated in oras a solid substrate, which may be glass, plastic, or other solidpolymeric materials, and typically have a planar format for ease ofdetecting and monitoring sample and reagent movement, especially viaoptical or electrochemical methods. Features of a microfluidic deviceusually have cross-sectional dimensions of less than a few hundredsquare micrometers and passages typically have capillary dimensions,e.g. having maximal cross-sectional dimensions of from about 500 micro mto about 0.1 micro m. Microfluidics devices typically have volumecapacities in the range of from 1 micro L to a few nL, e.g. 10-100 mL.The fabrication and operation of microfluidics devices are well-known inthe art as exemplified by the following references that are incorporatedby reference: Ramsey, U.S. Pat. Nos. 6,001,229; 5,858,195; 6,010,607;and 6,033,546; Soane et al, U.S. Pat. Nos. 5,126,022 and 6,054,034;Nelson et al, U.S. Pat. No. 6,613,525; Maher et al, U.S. Pat. No.6,399,952; Ricco et al, International patent publication WO 02/24322;Bjornson et al, International patent publication WO 99/19717; Wilding etal, U.S. Pat. Nos. 5,587,128; 5,498,392; Sia et al, Electrophoresis, 24:3563-3576 (2003); Unger et al, Science, 288: 113-116 (2000); Enzelbergeret al, U.S. Pat. No. 6,960,437.

“Nucleic acid sequence-based amplification” or “NASBA” is anamplification reaction based on the simultaneous activity of a reversetranscriptase (usually avian myeloblastosis virus (AMV) reversetranscriptase), an RNase H, and an RNA polymerase (usually T7 RNApolymerase) that uses two oligonucleotide primers, and which underconventional conditions can amplify a target sequence by a factor in therange of 109 to 1012 in 90 to 120 minutes. In a NASBA reaction, nucleicacids are a template for the amplification reaction only if they aresingle stranded and contain a primer binding site. Because NASBA isisothermal (usually carried out at 41 degrees centigrade with the aboveenzymes), specific amplification of single stranded RNA may beaccomplished if denaturation of double stranded DNA is prevented in thesample preparation procedure. That is, it is possible to detect a singlestranded RNA target in a double stranded DNA background without gettingfalse positive results caused by complex genomic DNA, in contrast withother techniques, such as RT-PCR. By using fluorescent indicatorscompatible with the reaction, such as molecular beacons, NASBAs may becarried out with real-time detection of the amplicon. Molecular beaconsare stem-and-loop-structured oligonucleotides with a fluorescent labelat one end and a quencher at the other end, e.g. 5′-fluorescein and3′-(4-(dimethylamino)phenyl)azo)benzoic acid (i.e., 3′-DABCYL), asdisclosed by Tyagi and Kramer (cited above). An exemplary molecularbeacon may have complementary stem strands of six nucleotides, e.g. 4G's or C's and 2 A's or T's, and a target-specific loop of about 20nucleotides, so that the molecular beacon can form a stable hybrid witha target sequence at reaction temperature, e.g. 41 degrees centigrade Atypical NASBA reaction mix is 80 mM Tris-HCl [pH 8.5], 24 mM MgCl2, 140mM KCl, 1.0 mM DTT, 2.0 mM of each dNTP, 4.0 mM each of ATP, UTP andCTP, 3.0 mM GTP, and 1.0 mM ITP in 30 percent DMSO. Primer concentrationis 0.1 micro M and molecular beacon concentration is 40 nM. Enzyme mixis 375 sorbitol, 2.1 micro g BSA, 0.08 U RNase H, 32 U T7 RNApolymerase, and 6.4 U AMV reverse transcriptase. A reaction may comprise5 micro L sample, 10 micro L NASBA reaction mix, and 5 micro L enzymemix, for a total reaction volume of 20 micro L. Further guidance forcarrying out real-time NASBA reactions is disclosed in the followingreferences that are incorporated by reference: Polstra et al, BMCInfectious Diseases, 2: 18 (2002); Leone et al, Nucleic Acids Research,26: 2150-2155 (1998); Gulliksen et al, Anal. Chem., 76: 9-14 (2004);Weusten et al, Nucleic Acids Research, 30(6) e26 (2002); Deiman et al,Mol. Biotechnol., 20: 163-179 (2002). Nested NASBA reactions are carriedout similarly to nested PCRs; namely, the amplicon of a first NASBAreaction becomes the sample for a second NASBA reaction using a new setof primers, at least one of which binds to an interior location of thefirst amplicon.

“Polymerase chain reaction,” or “PCR,” means a reaction for the in vitroamplification of specific DNA sequences by the simultaneous primerextension of complementary strands of DNA. In other words, PCR is areaction for making multiple copies or replicates of a target nucleicacid flanked by primer binding sites, such reaction comprising one ormore repetitions of the following steps: (i) denaturing the targetnucleic acid, (ii) annealing primers to the primer binding sites, and(iii) extending the primers by a nucleic acid polymerase in the presenceof nucleoside triphosphates. Usually, the reaction is cycled throughdifferent temperatures optimized for each step in a thermal cyclerinstrument. Particular temperatures, durations at each step, and ratesof change between steps depend on many factors well-known to those ofordinary skill in the art, e.g. exemplified by the references: McPhersonet al, editors, PCR: A Practical Approach and PCR2: A Practical Approach(IRL Press, Oxford, 1991 and 1995, respectively). For example, in aconventional PCR using Taq DNA polymerase, a double stranded targetnucleic acid may be denatured at a temperature>90 degrees centigrade,primers annealed at a temperature in the range 50-75 degrees centigrade,and primers extended at a temperature in the range 72-78 degreescentigrade The term “PCR” encompasses derivative forms of the reaction,including but not limited to, RT-PCR, real-time PCR, nested PCR,quantitative PCR, multiplexed PCR, and the like. Reaction volumes rangefrom a few hundred nanoliters, e.g. 200 mL, to a few hundred micro L,e.g. 200 micro L. “Reverse transcription PCR,” or “RT-PCR,” means a PCRthat is preceded by a reverse transcription reaction that converts atarget RNA to a complementary single stranded DNA, which is thenamplified, e.g. Tecott et al, U.S. Pat. No. 5,168,038, which patent isincorporated herein by reference. “Real-time PCR” means a PCR for whichthe amount of reaction product, i.e. amplicon, is monitored as thereaction proceeds. There are many forms of real-time PCR that differmainly in the detection chemistries used for monitoring the reactionproduct, e.g. Gelfand et al, U.S. Pat. No. 5,210,015 (“taqman”); Wittweret al, U.S. Pat. Nos. 6,174,670 and 6,569,627 (intercalating dyes);Tyagi et al, U.S. Pat. No. 5,925,517 (molecular beacons); which patentsare incorporated herein by reference. Detection chemistries forreal-time PCR are reviewed in Mackay et al, Nucleic Acids Research, 30:1292-1305 (2002), which is also incorporated herein by reference.“Nested PCR” means a two-stage PCR wherein the amplicon of a first PCRbecomes the sample for a second PCR using a new set of primers, at leastone of which binds to an interior location of the first amplicon. Asused herein, “initial primers” in reference to a nested amplificationreaction mean the primers used to generate a first amplicon, and“secondary primers” mean the one or more primers used to generate asecond, or nested, amplicon. “Multiplexed PCR” means a PCR whereinmultiple target sequences (or a single target sequence and one or morereference sequences) are simultaneously carried out in the same reactionmixture, e.g. Bernard et al, Anal. Biochem., 273: 221-228 (1999)(two-color real-time PCR). Usually, distinct sets of primers areemployed for each sequence being amplified. Typically, the number oftarget sequences in a multiplex PCR is in the range of from 2 to 10, orfrom 2 to 6, or more typically, from 2 to 4.

“Quantitative PCR” means a PCR designed to measure the abundance of oneor more specific target sequences in a sample or specimen. QuantitativePCR includes both absolute quantitation and relative quantitation ofsuch target sequences. Quantitative measurements are made using one ormore reference sequences that may be assayed separately or together witha target sequence. The reference sequence may be endogenous or exogenousto a sample or specimen, and in the latter case, may comprise one ormore competitor templates. Typical endogenous reference sequencesinclude segments of transcripts of the following genes: beta-actin,GAPDH, beta 2-microglobulin, ribosomal RNA, and the like. Techniques forquantitative PCR are well-known to those of ordinary skill in the art,as exemplified in the following references that are incorporated byreference: Freeman et al, Biotechniques, 26: 112-126 (1999);Becker-Andre et al, Nucleic Acids Research, 17: 9437-9447 (1989);Zimmerman et al, Biotechniques, 21: 268-279 (1996); Diviacco et al,Gene, 122: 3013-3020 (1992); Becker-Andre et al, Nucleic Acids Research,17: 9437-9446 (1989); and the like.

“Polynucleotide” and “oligonucleotide” are used interchangeably and eachmeans a linear polymer of nucleotide monomers. Monomers making uppolynucleotides and oligonucleotides are capable of specifically bindingto a natural polynucleotide by way of a regular pattern ofmonomer-to-monomer interactions, such as Watson-Crick type of basepairing, base stacking, Hoogsteen or reverse Hoogsteen types of basepairing, or the like. Such monomers and their internucleosidic linkagesmay be naturally occurring or may be analogs thereof, e.g. naturallyoccurring or non-naturally occurring analogs. Non-naturally occurringanalogs may include PNAs, phosphorothioate internucleosidic linkages,bases containing linking groups permitting the attachment of labels,such as fluorophores, or haptens, and the like. Whenever the use of anoligonucleotide or polynucleotide requires enzymatic processing, such asextension by a polymerase, ligation by a ligase, or the like, one ofordinary skill would understand that oligonucleotides or polynucleotidesin those instances would not contain certain analogs of internucleosidiclinkages, sugar moieties, or bases at any or some positions.Polynucleotides typically range in size from a few monomeric units, e.g.5-40, when they are usually referred to as “oligonucleotides,” toseveral thousand monomeric units. Whenever a polynucleotide oroligonucleotide is represented by a sequence of letters (upper or lowercase), such as “ATGCCTG,” it will be understood that the nucleotides arein 5′-3′ order from left to right and that “A” denotes deoxyadenosine,“C” denotes deoxycytidine, “G” denotes deoxyguanosine, and “T” denotesthymidine, “I” denotes deoxyinosine, “U” denotes uridine, unlessotherwise indicated or obvious from context. Unless otherwise noted theterminology and atom numbering conventions will follow those disclosedin Strachan and Read, Human Molecular Genetics 2 (Wiley-Liss, New York,1999). Usually polynucleotides comprise the four natural nucleosides(e.g. deoxyadenosine, deoxycytidine, deoxyguanosine, deoxythymidine forDNA or their ribose counterparts for RNA) linked by phosphodiesterlinkages; however, they may also comprise non-natural nucleotideanalogs, e.g. including modified bases, sugars, or internucleosidiclinkages. It is clear to those skilled in the art that where an enzymehas specific oligonucleotide or polynucleotide substrate requirementsfor activity, e.g. single stranded DNA, RNA/DNA duplex, or the like,then selection of appropriate composition for the oligonucleotide orpolynucleotide substrates is well within the knowledge of one ofordinary skill, especially with guidance from treatises, such asSambrook et al, Molecular Cloning, Second Edition (Cold Spring HarborLaboratory, New York, 1989), and like references.

“Primer” means an oligonucleotide, either natural or synthetic that iscapable, upon forming a duplex with a polynucleotide template, of actingas a point of initiation of nucleic acid synthesis and being extendedfrom its 3′ end along the template so that an extended duplex is formed.Extension of a primer is usually carried out with a nucleic acidpolymerase, such as a DNA or RNA polymerase. The sequence of nucleotidesadded in the extension process is determined by the sequence of thetemplate polynucleotide. Usually primers are extended by a DNApolymerase. Primers usually have a length in the range of from 14 to 40nucleotides, or in the range of from 18 to 36 nucleotides. Primers areemployed in a variety of nucleic amplification reactions, for example,linear amplification reactions using a single primer, or polymerasechain reactions, employing two or more primers. Guidance for selectingthe lengths and sequences of primers for particular applications is wellknown to those of ordinary skill in the art, as evidenced by thefollowing references that are incorporated by reference: Dieffenbach,editor, PCR Primer: A Laboratory Manual, 2nd Edition (Cold Spring HarborPress, New York, 2003).

“Readout” means a parameter, or parameters, which are measured and/ordetected that can be converted to a number or value. In some contexts,readout may refer to an actual numerical representation of suchcollected or recorded data. For example, a readout of fluorescentintensity signals from a microarray is the address and fluorescenceintensity of a signal being generated at each hybridization site of themicroarray; thus, such a readout may be registered or stored in variousways, for example, as an image of the microarray, as a table of numbers,or the like. Likewise, a readout of a real-time PCR can be one or morefluorescent intensity signals within specified frequency bands asfunctions of time, or other reaction parameter related to time.

“Specific” or “specificity” in reference to the binding of one moleculeto another molecule, such as a labeled target sequence for a probe,means the recognition, contact, and formation of a stable complexbetween the two molecules, together with substantially less recognition,contact, or complex formation of that molecule with other molecules. Inone aspect, “specific” in reference to the binding of a first moleculeto a second molecule means that to the extent the first moleculerecognizes and forms a complex with another molecule in a reaction orsample, it forms the largest number of the complexes with the secondmolecule. Preferably, this largest number is at least fifty percent.Generally, molecules involved in a specific binding event have areas ontheir surfaces or in cavities giving rise to specific recognitionbetween the molecules binding to each other. Examples of specificbinding include antibody-antigen interactions, enzyme-substrateinteractions, formation of duplexes or triplexes among polynucleotidesand/or oligonucleotides, receptor-ligand interactions, and the like. Asused herein, “contact” in reference to specificity or specific bindingmeans two molecules are close enough that weak noncovalent chemicalinteractions, such as Van der Waal forces, hydrogen bonding,base-stacking interactions, ionic and hydrophobic interactions, and thelike, dominate the interaction of the molecules.

“Tm” or “melting temperature” means the temperature at which apopulation of double-stranded nucleic acid molecules becomes halfdissociated into single strands. Several equations for calculating theTm of nucleic acids are well known in the art. For example, a simpleestimate of the Tm value may be calculated by the equation. Tm=81.5+0.41(percent G+C), when a nucleic acid is in aqueous solution at 1 M NaCl.Methods for calculating Tm based on more complete models of duplexformation and dissociation are found in Breslauer et al, Proc. Natl.Acad. Sci., 83: 3746-3750 (1986); and Wetmur, Crit. Rev. Biochem. Mol.Biol., 26: 227-259 (1991).

“Sample” means a quantity of material from a biological, environmental,medical, or patient source in which detection or measurement of targetnucleic acids is sought. On the one hand it is meant to include aspecimen or culture (e.g., microbiological cultures). On the other hand,it is meant to include both biological and environmental samples. Asample may include a specimen of synthetic origin. Biological samplesmay be animal, including human, fluid, solid (e.g., stool) or tissue, aswell as liquid and solid food and feed products and ingredients such asdairy items, vegetables, meat and meat by-products, and waste.Biological samples may include materials taken from a patient including,but not limited to cultures, blood, saliva, cerebral spinal fluid,pleural fluid, milk, lymph, sputum, semen, needle aspirates, and thelike. Biological samples may be obtained from all of the variousfamilies of domestic animals, as well as feral or wild animals,including, but not limited to, such animals as ungulates, bear, fish,rodents, etc. Environmental samples include environmental material suchas surface matter, soil, water and industrial samples, as well assamples obtained from food and dairy processing instruments, apparatus,equipment, utensils, disposable and non-disposable items. These examplesare not to be construed as limiting the sample types applicable to thepresent invention. The terms “sample” and “specimen” are usedinterchangeably.

Detailed Description of the Invention

The practice of the present invention may employ, unless otherwiseindicated, conventional techniques and descriptions of organicchemistry, polymer technology, molecular biology (including recombinanttechniques), cell biology, biochemistry, and analytical instrumentation,which are within the skill of the art. Such conventional techniquesinclude fluorescence measurement, optical signal collection,instrumentation control, data analysis, electronics, mechanicalengineering, fluid handling, and the like. Specific illustrations ofsuitable techniques can be had by reference to the example herein below.However, other equivalent conventional procedures can, of course, alsobe used. Such conventional techniques and descriptions can be found instandard laboratory manuals such as Genome Analysis: A Laboratory ManualSeries (Vols. I-IV), Using Antibodies: A Laboratory Manual, Cells: ALaboratory Manual, PCR Primer: A Laboratory Manual, and MolecularCloning: A Laboratory Manual (all from Cold Spring Harbor LaboratoryPress), as well as other treatises and guides cited below.

The invention is directed to reagent reservoir systems and disposablereaction cassettes employing such systems. The invention addressesproblems associated with the storage and activation of assay reagents,particularly proteins, which have been at least partially dehydrated andembedded in a protective matrix so that solid or semi-solid precipitatesor particles are formed. Such solid and semi-solid masses in particulateor powder form often have physical properties, such as electrostaticcharge, that make them difficult to control, since the charge-carryingparticulates may be attracted to regions of a vessel or reservoir wherethere is no contact with an activating liquid (e.g., a solution orbuffer). In one aspect, such problems are overcome by providing areagent reservoir system comprising a chamber having an elongated shapeand containing a retaining member (preferably buoyant) that obstructspassage of reagent-containing solids or semi-solids, referred to hereinas “lyophilized particulates,” thereby restricting their dispersalthrough the chamber. That is, the retaining member prevents the driedreagent, but neither gases nor liquids, from entering the region betweena first end of the chamber and the retaining member. The chamber furthercomprises an exhaust port at the first end and a fluid port at the endof the chamber containing the dried reagent (i.e., a second end). Thefirst end with the exhaust port, the retaining member and the second endwith the fluid port and containing dried reagent are disposed along thelongitudinal axis of the chamber.

In a preferred embodiment, the retaining member floats, or is buoyant,in whatever activation liquid is employed to hydrate and/or activate thedried reagent. In one aspect, such buoyancy is obtained by making thebuoyant retaining member from a material having a lower specific gravitythan that of the activation liquid; however, it may also be aheterogeneous structure having an average specific gravity less thanthat of the activation liquid. Preferably, the buoyancy of the retainingmember in the activation liquid is selected so that 75 to 90 percent ofthe volume of the buoyant retaining member is submerged. Optionally, achamber also contains a fixed retaining member that is disposed in theregion of the chamber to the side of the buoyant retaining memberopposite that occupied by the dried reagent. The function of the fixedretaining member includes obstructing passage of the buoyant retainingmember (but not the passage of liquids or gases). The fixed retainingmember may be fabricated as an integral piece of the chamber of it canbe a separate piece that is placed in the chamber during assembly. Ineither case, placement of the fixed retaining member allows one tocontrol the extent of travel of the buoyant retaining member inside ofthe chamber. When a fixed retaining member is employed, the buoyantretaining member further prevents dried reagent (e.g., lyophilizedparticulates) from (i) coming into contact with (and sticking to orprecipitating on) crevices or edges of the fixed retaining member byfoaming action in the activation liquid, and (ii) becoming trapped inthe surface tension of the activation liquid so that incompletesolvation occurs. Preferably, the fixed retaining member is disposed inthe chamber at a position that permits the formation of a gap between itand the buoyant retaining member and lyophilized particulates wheneverthe longitudinal axis of the chamber is in a vertical orientation (inthe absence of liquid).

As discussed more fully below, the invention further provides disposablereaction cassettes that incorporate one or more reagent reservoirsystems.

The above principles of the invention are illustrated by the embodimentsshown in FIGS. 1A-1C. FIG. 1A illustrates the operation of an embodimentthat does not include a fixed retaining member and FIGS. 1B and 1Cillustrate two embodiments that include two different kinds of fixedretaining members. In FIG. 1A, elongated chamber (100) havinglongitudinal axis (101) is shown in a vertical orientation with firstend (102) at the top and second end (104) at the bottom, which is thepreferred orientation whenever gravity or positive or negative pressureis used to force liquid in or out of chamber (100). Buoyant retainingmember (105) rests at second end (104) on top of dried reagent, shown aslyophilized particulates (108), preventing their dispersal to first end(102). The inside diameter of chamber (100), diameter and/or shape ofbuoyant retaining member (105), and the diameter and/or shape oflyophilized particulates (108) are selected so that gaps (107) betweenthe inner wall of chamber (100) and outer surface of buoyant retainingmember (105) do not allow passage of lyophilized particulates (108),particularly prior to transfer of liquid to chamber (100). In oneaspect, the chamber, buoyant retaining member, and fixed retainingmember are made of a suitable plastic or polymeric solid, such aspolystyrene, polyvinylchloride, polycarbonate, polypropylene,polyethylene, or any other plastic that is rigid and inert with respectto the fluids and reagents used with the system.

Exhaust port (110) is located at first end (102) and fluid port (106) islocated at second end (104). Activation liquid (114) is transferredthrough passage (112) and fluid port (106) to the interior of chamber(100) thereby floating buoyant retaining member (105). Activation liquid(114) is typically an aqueous solution or suspension or a buffer such aswater. In some embodiments, the liquid contains the analyte (e.g.,nucleic acid) to be tested with the reagent(s) stored in the chamber.Gas displaced by activation liquid (114) escapes (116) through exhaustport (110). Preferably, exhaust port (110) comprises a gas permeable andliquid impermeable membrane, such as a woven polyolefin film, forexample, Tyrek® film (DuPont), or the like. Lyophilized particulates(108) dissolve (118) in activation liquid (114) to form a functionalreagent for an assay, while a predetermined amount of activation liquid(114) is loaded into chamber (100), and optionally during apredetermined incubation period thereafter. The latter predeterminedincubation time depends on the nature and solubility properties of thereagents and other compounds making up lyophilized particulates (108).Chamber (100) could also be subjected to heating and mixing steps toassist the dissolution of lyophilized particulates (108). Once afunctional reagent solution is formed, it is removed (122) from chamber(100) through fluid port (106) for use in an assay. Movement of liquidsin and out of chamber (100) can be accomplished in many different wayswell known to those of ordinary skill in the art, such increasing ordecreasing pressure on the liquid by a pump, driving liquid out of thechamber by increasing gas pressure by way of exhaust port (110), usinggravity to drain the liquid, and the like. In other embodiments, morethan one fluid port can be used. For example, separate inlet and outletports may be used, and in such cases, one of the ports may be located atfirst end (102), or at other locations in chamber (100).

Although the preferred embodiment includes a buoyant retaining member,alternative embodiments may employ a non-buoyant retaining member(having a density greater than the activation liquid). In thesealternative embodiments, when the liquid is added to the chamber, theretaining member sinks to the bottom of the chamber when the driedreagent dissolves in the liquid. In these alternative embodiments, thefluid port may be offset from the bottom of the chamber to prevent anyproblems in removing the reagent solution from the chamber (e.g., sothat the non-buoyant retaining member does not block the port).

In a preferred embodiment, the reagent reservoir system furthercomprises a fixed retainer member disposed along longitudinal axis (101)between first end (102) and buoyant retaining member (105). As mentionedabove, a fixed retaining member can be implemented in many ways forcarrying out its primary function of obstructing passage of buoyantretaining member (105) without obstructing passage of either gases orliquids. The implementation selected depends on conventional factors ofinstrument design, including cost of materials, ease of manufacture,ease of assembly, and the like. FIGS. 1B and 1C illustrate two exemplaryembodiments of fixed retainer members. In FIG. 1B, fixed retainingmember (130) has a hexagonal cross section and is closely fitted intothe interior of chamber (100) so that it is immobilized by contact ofedges (132) with the inner wall of chamber (100). Between edges (132)there are gaps that allow passage (136) of gas or liquid. Alternatively,a chamber (100) can be provided that has a transverse cross section(142) that is not circular, so that a spherically shaped fixed retainingmember (144) can be fitted, or wedged, into the interior of chamber(100) and immobilized by contacts (146) with the inner wall of chamber(100). Since cross section of chamber (100) is not circular, gaps arepresent for gas or liquid to pass (148).

In one aspect, the invention provides disposable reaction cassettes thatemploy one or more reagent reservoir systems described above. Suchreaction cassettes can be designed to perform many different kinds ofassays and are typically used with an apparatus that provides ancillaryfunctions, such as temperature control, detection systems, mechanical orelectrical power sources to pump, or otherwise move, liquids into andout of cassettes, and the like. Such reaction cassettes can vary widelyis shape and scale, from macro-scale, e.g. reaction volumes, reservoirvolumes, sample volumes, in 1-10 mL range, to micro-scale, e.g. reactionvolumes, reservoir volumes, sample volumes in nanoliter to microliterrange. Exemplary systems that may be used with reagent reservoir systemsof the invention include fluidly closed reaction systems employing arotary valve and a piston-type fluid pump under microprocessor control,such as disclosed in Christel et al, U.S. Pat. No. 6,369,893 and Dority,U.S. Pat. No. 6,374,684; and microfluidics devices, such as disclosed inthe references cited under Definitions, and further disclosed in Shojiet al, Appl. Biochem. Biotechnol., 41: 21-34 (1993) and J. Micromech.Microeng., 4: 157-171 (1994); McCormick et al, Anal. Chem., 69:2626-2630 (1997); Cheng et al, Topics Curr. Chem., 194: 215-231 (1998);Stave et al, U.S. Pat. No. 6,663,833; Neri et al, U.S. Pat. No.5,714,380; Northrup et al, U.S. Pat. No. 5,589,136; and the like.Additional reaction cassette system that can be used with the presentinvention include Petersen et al, U.S. patent publication 2005/0042137;and Taylor et al, U.S. patent publication 2004/0166031; Catanzariti etal, U.S. Pat. No. 5,786,182; and Itoh et al, U.S. patent publication2005/0180880; which are incorporated by reference. Such systems arecapable of fluidly transferring reactants, samples, and reactionproducts between reservoirs and reaction chambers in a controlledmanner. That is, such systems move reactants, samples, reactionproducts, and the like, in liquid solutions under liquid-moving force ina directed manner. Liquid-moving forces include differential pressuregenerated by various kinds of pumps or compressed gas reservoirs,electrokinetic pumps, and the like.

In one aspect, disposable reaction cassettes of the invention may beconveniently implemented by specific designs and methods of operation ofrotary valves, reactant and waste reservoirs, and reaction chambersgenerally disclosed in Dority (cited above). In another aspect, in whichreal-time monitoring of amplification reactions is desired, suchapparatus is conveniently used with the temperature controller andfluorometer disclosed by Christel et al (cited above).

FIG. 2A shows diagrammatically a disposable reaction cassette thatfollows the general design approach disclosed in Dority (cited above)for carrying out amplification reactions to amplify targetpolynucleotides from a sample under fluidly closed conditions. Asmentioned above, such a disposable reaction cassette may be used with adetection apparatus disclosed by Christel et al (cited above) andillustrated diagrammatically in FIG. 2B. After a sample or specimen isloaded into the reaction cassette and pre-conditioned, e.g. bydisrupting tissue, lysing cells, and the like, the resulting solution isfluidly transferred to a sample reservoir from which one or moreportions are dispensed to a reagent reservoir system under programmedcontrol for mixing with amplification reagents to form a reactionmixture. The reaction mixture is then fluidly transferred to a reactionchamber where an amplification reaction takes place.

FIG. 2A shows housing (200) that contains rotary valve (202) havinginternal chamber (204) that is operationally connected to piston-typepump (206). Up-strokes of piston (256) of pump (206) pressurize chamber(204) and force fluid contents out through whatever ports that may be incommunication with reservoirs or the like; likewise, down strokes ofpiston (256) of pump (206) depressurize chamber (204) and draw fluids inthrough whatever ports may be open and in communication with reservoirsor the like. Further descriptions of the operation and construction ofsuch pump-rotary valve devices and the use of chamber (204) for samplepreparation is provided by Dority (cited above), which is incorporatedby reference for this purpose. Rotary valve (202) has various ports, forexample (250) and (252), and associated passages, (208) and (212), thatpermit chamber (204) to be in fluid communication with variousreservoirs (described more fully below) or reaction chamber (242)whenever such ports are aligned with corresponding ports to passages tosuch reservoirs or reaction chamber (242). In the present exemplaryembodiments, the longitudinal axes of such associated passages areradially disposed in rotary valve (202) within either one of two planesperpendicular to the axis of rotary valve (202) (shown with dashed lines(248) and (249)), such that chamber (204) may be placed in fluidcommunication with ports of passages to reservoirs, and the like,disposed in housing (200). Rotary valve (202) further includesconnecting passages (210), (211), and (213), which permit a port in oneplane of the valve to be placed in fluid communication with ports ofhousing (200) that are in the other plane of rotary valve (202). Suchconnection passages do not permit fluid communication with interiorchamber (204). As illustrated in FIG. 2A, when such connecting passagesare aligned at (246) with ports of passages (244) and (216), passages(244) and (216) are in fluid communication. Likewise, when suchconnecting passages are aligned at (238) with ports of passages (240)and (236), passages (240) and (236) are in fluid communication. In FIG.2A, cross-hatched passages and reservoirs in housing (200) are in thepump-proximal plane of rotary valve (202) (illustrated in the bottompanel of FIG. 2A by dotted line (249)), whereas the non-hatched passagesand reservoirs are in the pump-distal plane (illustrated in the bottompanel of FIG. 2A by dotted line (248)). As mentioned above, rotary valve(202) may place interior chamber (204) in fluid communication withvarious reservoirs and reaction chamber (242) that are connected bypassages and have ports in the seat of housing (200) that rotary valve(202) rotates within. In the present example, such reservoirs includethe following; (i) reagent reservoir system (214) containing firstamplification reagents, which may be fluidly connected to rotary valve(202) by passage (216); (ii) reservoir (218) containing lysing reagents,for example, for disrupting surface membranes of cellular samples, whichreservoir may be fluidly connected to rotary valve (202) by passage(220); (iii) sample reservoir (222) containing sample or specimenmaterial, which may be fluidly connected to rotary valve (202) bypassage (224); (iv) reservoir (226) containing wash solution, or washreagent, which may be fluidly connected to rotary valve (202) by passage(228); (v) waste reservoir (230), which may be fluidly connected torotary valve (202) by passage (232); and (vi) reagent reservoir system(234) containing second amplification reagents, which may be fluidlyconnected to rotary valve (202) by passage (236). It should be clearfrom the above example that design of rotary valve (202), e.g. selectionof the number and kind of passages, and the selection of the number andtype of reagent reservoirs in housing (200) is a matter of routinedesign choice of one of ordinary skill in the art.

A variety of instrumentation systems may be employed to implement assayreactions in disposable reaction cassettes so that an optical signal isgenerated related to one or more reaction parameters, such as ampliconconcentration. As described more fully below, in one aspect, amultichannel optical detection system disclosed by Christel et al, U.S.Pat. No. 6,369,893 is well-suited for such measurements. A schematic ofsuch a system applicable to the present invention is illustrated in FIG.2C. Christel et al provide diode lasers (2050) through (2056) forilluminating a reaction mixture in reaction chamber (2070). Fluorescenceexcited by laser diodes (2050) through (2056) is collected by detectors(2060) through (2066), which typically are each operationally associatedwith a bandpass filter that restricts the wavelength of light that isdetected. The excitation beams of laser diodes (2050) through (2056) maybe the same or different. In one aspect, bandpass filters are selectedto selectively pass fluorescence emitted by a plurality of spectrallyresolvable fluorescent dyes so that each detector (2060) through (2066)collects fluorescence primarily from only one of the plurality offluorescent dyes. For use with the present invention, one of the laserdiode-detector pairs, for example (2052) and (2062), is allocated todetecting the fluorescent signal from an amplicon corresponding to atarget polynucleotide, and one of the laser diode-detector pairs, forexample (2056) and (2066), is allocated to detecting fluorescent signalfrom an amplicon corresponding to a reference sequence.

Control of all components of the detection system and fluidly closedreaction system (2086) are controlled by microprocessor (2080). Opticalsignals collected by detectors (2060) through (2066) are processed byconventional optics and converted into electrical signals, which, afterconventional pre-amplification and conditioning (2082), are digitizedfor storage and/or further processing by microprocessor (2080). In oneaspect of the invention, microprocessor (2080) is programmed tocontinuously monitor the value of the signal collected by one of thedetectors, such as detector (2062). When the value reaches or exceeds apredetermined level, then microprocessor (2080) initiates a subroutinethat provides controllers (2084) with a series of commands to actuatecomponents of fluidly closed reaction system (2086) to initiate asubsequent step in the assay being conducted. Microprocessor (2080) alsochanges and/or regulates the temperature of reaction chamber (2070)through controller (2088). In embodiments employing closed-loop control,microprocessor (2080) may calculate values of characteristics ofintensity versus time curves at predetermined intervals so that they maybe compared to a predetermined level. When such calculated value reachesor exceeds a predetermined level, then microprocessor (2080) initiatesthe subroutine to start a subsequent assay step.

Stored Reagents and Assays

The nature, composition, and method of producing dried compounds forstoring assay reagents (referred to herein as “dried reagent”) varywidely and the formulation and production of such materials iswell-known to those of ordinary skill in the art as evidenced by thefollowing references that are incorporated by reference: Franks et al,U.S. Pat. No. 5,098,893; Cole, U.S. Pat. No. 5,102,788; Shen et al, U.S.Pat. No. 5,556,771; Treml et al, U.S. Pat. No. 5,763,157; De Rosier etal, U.S. Pat. No. 6,294,365; Buhl et al, U.S. Pat. No. 5,413,732;McMillan, U.S. patent publication 2006/0068398; McMillan et al, U.S.patent publication 2006/0068399; Schwegman et al (2005), Pharm. Dev.Technol., 10: 151-173; Nail et al (2002), Pharm. Biotechnol., 14:281-360; and the like. Dried reagents include, but are not limited to,solid and/or semi-solid particulates, powders, tablets, crystals,capsules, beads, spheres and the like, that are manufactured in avariety of ways. In one aspect, dried reagent comprises one or morelyophilized particulates such as reagent beads or spheres.

Lyophilized particulates may have uniform compositions, wherein eachparticulate has the same composition, or they may have differentcompositions, such that two or more different kinds of lyophilizedparticulates having different compositions are mixed together.Lyophilized particulates can contain reagents for all or part of a widevariety of assays and biochemical reactions, including immunoassays,enzyme-based assays, enzyme substrate assays, DNA sequencing reactions,and the like. Of particular interest are assays that involve theamplification of nucleic acids, i.e. the production of amplicons,including PCRs, NASBAs, rolling circle reactions, ligase-basedreactions, and the like. In one aspect, a lyophilized particulate of theinvention comprises an excipient and at least one reagent of an assay.

Lyophilized particulates may be manufactured in predetermined sizes andshapes, which may be determined by the type of assay being conducted,desired reaction volume, desired speed of dissolution, and the like. Inone embodiment, for example in devices designed in accordance withDority (cited above), lyophilized particulates have a spherical shapehaving a diameter in the range of form 0.5 to 5 mm; that is, thelyophilized particulates are in the form of beads. Excipients areusually inert substances added to a material in order to confer asuitable consistency or form to the material. A large number ofexcipients are known to those of skill in the art and can comprise anumber of different chemical structures. Examples of excipients, whichmay be used in the present invention, include carbohydrates, such assucrose, glucose, trehalose, melezitose, dextran, and mannitol; proteinssuch as BSA, gelatin, and collagen; and polymers such as PEG andpolyvinyl pyrrolidone (PVP). The total amount of excipient in thelyophilized bead may comprise either single or multiple compounds. Insome embodiments, the type of excipient is a factor in controlling theamount of bead hygroscopy. Lowering bead hygroscopy can enhance thebead's integrity (accuracy of weighing beads) and cryoprotectantabilities. However, removing all water from the bead would havedeleterious effects on those reaction components, proteins for example,that require certain amounts of bound water in order to maintain properconformations. In general, the excipient level in the beads should beadjusted to allow moisture levels of less than 3 percent. In someembodiments, the excipient is trehalose, mannitol, dextran, orcombinations thereof. The amount of excipient is also a factor incontrolling the amount of bead hygroscopy. There are limits to theamount of excipient which can be added to form a bead. If the amount ofexcipient is too low, the material does not coalesce to form a bead-likeshape. At the high end, excipient amounts are limited by the solubilityof the excipient in the bead buffer formulation. The amount is alsodependent upon the properties of the excipient. In an exemplaryembodiment, trehalose is present from between 5 percent to 20 percent(w/v). In another exemplary embodiment, mannitol is present from between2 percent to 20 percent (w/v). In yet another exemplary embodiment,mannitol is present from between 2 percent to 20 percent (w/v) anddextran is present from between 0.5 percent to 5 percent (w/v). In stillanother exemplary embodiment, mannitol is present in the lyophilizedbead in a weight percentage of between 40 percent to 75 percent (w/w).The symbol “w/w” refers to the dry weight of the excipient divided bythe dry weight of the lyophilized bead. The symbol “w/v” refers to thedry weight (in grams) of the excipient divided by the volume (in 100 mL)of the bead buffer formulation.

As mentioned above, beads or lyophilized particulates containingreagents for nucleic acid amplification reactions are of specialinterest, particularly reagents for PCR. In some embodiments,lyophilized particulates for PCR may comprise a mixture of compositionssuch that one set of lyophilized particulates, or beads, comprises abuffer, excipient, a carrier protein, magnesium, and an antifoam agent,and another set of beads may comprise generic assay components, such asa DNA polymerase (Taq polymerase, polymerases complexed with hot startantibodies such as Platinum polymerases (Invitrogen, Carlsbad, Calif.)),RNA polymerase, reverse transcriptase, and/or deoxynucleosidetriphosphates (e.g., dATP, dCTP, dTTP, dGTP), and yet another set ofbeads may comprise specific assay components, such as primers whichcorrespond a particular DNA sequence of interest, as well as probeswhich will detect the presence of primer hybridization with the DNAsequence of interest.

By way of example, lyophilized particulates for carrying out a PCRcomprise the following reagents: (1) Oligonucleotide Primers. Theoligonucleotides that are used in a PCR as well as oligonucleotidesdesigned to detect amplification products can be chemically synthesized.These oligonucleotides can be labeled with radioisotopes,chemiluminescent moieties, or fluorescent moieties. Such labels areuseful for the characterization and detection of amplification productsusing the methods and compositions of the present invention. The primercomponents may be present in the PCR reaction mixture at a concentrationof, e.g., between 0.1 and 1.0 micro M. The primer length can be between,e.g., 8-100 nucleotides in length. In order to aid in hybridization withthe nucleic acid sequence, the primers in some embodiments have 50-60percent G and C composition. In the choice of primer, it is preferableto have exactly matching bases at the 3′ end of the primer but thisrequirement decreases to relative insignificance at the 5′ end. In someembodiments, the primers of the invention all have approximately thesame melting temperature. (2) Buffer. Exemplary buffers that may beemployed, include, e.g., HEPES, borate, phosphate, carbonate, barbital,Tris, etc.-based buffers. See Rose et al., U.S. Pat. No. 5,508,178. ThepH of the reaction should be maintained in the range of about 4.5 toabout 9.5. See U.S. Pat. No. 5,508,178. The standard buffer used inamplification reactions is a Tris based buffer between 10 and 50 mM witha pH of around 8.3 to 8.8. One of skill in the art will recognize thatbuffer conditions should be designed to allow for the function of allreactions of interest. Thus, buffer conditions can be designed tosupport the amplification reaction as well as any enzymatic reactionsassociated with producing signals from probes. A particular reactionbuffer can be tested for its ability to support various reactions bytesting the reactions both individually and in combination. (3) SaltConcentration. The concentration of salt present in the reaction mixturecan affect the ability of primers to anneal to the target nucleic acid.Potassium chloride is typically added up to a concentration of about 50mM or more to the reaction mixture to promote primer annealing. Sodiumchloride can also be added to promote primer annealing. (4) MagnesiumIon Concentration. The concentration of magnesium ion in the reactioncan be critical to amplifying the desired sequence(s). Primer annealing,strand denaturation, amplification specificity, primer-dimer formation,and enzyme activity are all examples of parameters that are affected bymagnesium concentration. Amplification reactions can contain, e.g.,about a 0.5 to 2.5 mM magnesium concentration excess over theconcentration of dNTPs. The presence of magnesium chelators in thereaction can affect the optimal magnesium concentration. A series ofamplification reactions can be carried out over a range of magnesiumconcentrations to determine the optimal magnesium concentration. Theoptimal magnesium concentration can vary depending on the nature of thetarget nucleic acid(s) and the primers being used, among otherparameters. A common source of magnesium ion is MgCl2. (5) CarrierProteins. Carrier proteins useful in the present invention include butare not limited to albumin (e.g., bovine serum albumin) and gelatin. (6)Deoxynucleoside Triphosphate Concentration. Deoxynucleosidetriphosphates (dNTPs) are added to the reaction to a final concentrationof about 20 micro M to about 300 micro M. Each of the four dNTPs (G, A,C, T) are generally present at equivalent concentrations. (7) NucleicAcid Polymerase. A variety of DNA dependent polymerases are commerciallyavailable. For example, Taq DNA Polymerase may be used to amplify targetDNA sequences. The PCR assay may be carried out using as an enzymecomponent a source of thermostable DNA polymerase suitably comprisingTaq DNA polymerase which may be the native enzyme purified from Thermusaquaticus and/or a genetically engineered form of the enzyme. Othercommercially available polymerase enzymes include, e.g., Taq polymerasesmarketed by Promega or Pharmacia. Other examples of thermostable DNApolymerases that could be used in the invention include DNA polymerasesobtained from, e.g., Thermus and Pyrococcus species. Concentrationranges of the polymerase may range from 1-5 units per reaction mixture.The reaction mixture is typically between 20 and 100 micro L. In someembodiments, a “hot start” methodology polymerase can be used to preventextension of mis-priming events as the temperature of a reactioninitially increases. Hot starts are particularly useful in the contextof multiplex PCR. Examples of hot start methodologies include heatlabile adducts attached to a polymerase or ligase requiring a heatactivation step (typically 95 degrees centigrade for approximately 10-15minutes) or an antibody associated with the polymerase or ligase toprevent activation. Examples of different hot start methodologies areprovided in the following articles (Chou, et al., Nucleic Acids Research20: 1717-1723 (1992); Bassam, et al., Bio Techniques 14: 31-33 (1993);Horton, et al., Bio Techniques 16: 42-43 (1994); Kellogg, D. E., et al.,Bio techniques 16: 1134-1137 (1994); Birch D. E., et al., Nature 381:445-446 (1996); Bost, D. A., et al., The FASEB Journal 11: A1370(1997)), which are incorporated herein by reference. (8) Other Agents.Assorted other agents are sometimes added to the reaction to achieve thedesired results. For example, DMSO can be added to the reaction, thoughit is reported to inhibit the activity of Taq DNA Polymerase.Nevertheless, DMSO has been recommended for the amplification ofmultiple target sequences in the same reaction. Non-ionic detergents(e.g. Tween-20) can also be added to amplification reactions. Inaddition, methylisothiazolinone (MIT) can be added to the reactionmixture.

Lyophilized particulates may also include one or more internal standardsand/or one or more indicators, particularly fluorescent indicators, suchas molecular beacons, scorpion primers, sunrise primers, and the like.

What is claimed is:
 1. A method for dissolving a dried reagent in aliquid, the method comprising: a) providing a chamber for holding thedried reagent, the chamber having a longitudinal axis, a fluid port, anexhaust port, and a retaining member, wherein the dried reagent isdisposed in a reagent holding end of the chamber, wherein the fluid portis disposed at the reagent holding end of the chamber, and wherein theretaining member is disposed in the chamber between the exhaust port andthe dried reagent and is sized such that the space between the interiorof the chamber and the retaining member is smaller than the driedreagent, obstructing passage of the dried reagent from the reagentholding end toward the opposite end of the chamber; and b) transferringthe liquid through the fluid port to the reagent holding end of thechamber to dissolve the dried reagent in the liquid without the driedreagent passing the retaining member.
 2. The method of claim 1, whereinthe retaining member is fixed to the interior of the chamber.
 3. Themethod of claim 1, wherein the retaining member is movably disposed inthe chamber.
 4. The method of claim 3, wherein the chamber furthercomprises a fixed member immobilized within the chamber between theexhaust port and the retaining member, the fixed member obstructingpassage of the retaining member toward the exhaust port.
 5. The methodof claim 3, wherein the longitudinal axis of the chamber is in avertical orientation with the reagent holding end being the bottom endand the opposite end being the top end, and wherein the retaining memberis buoyant in the liquid.
 6. The method of claim 1, wherein each of thereagent chamber, the fixed member, and the retaining member is composedof a polymeric material.
 7. The method of claim 1, wherein the retainingmember has a density less than 1,000 kg/m³.
 8. The method of claim 1,wherein the dried reagent comprises an enzyme.
 9. The method of claim 1,wherein the dried reagent comprises one or more lyophilizedparticulates.
 10. The method of claim 1, wherein the dried reagentcomprises nucleic acid amplification reagents.
 11. The method of claim1, further comprising, subsequent to b), moving the liquid in and out ofthe chamber via the fluid port.
 12. A method for assembling a reactionmixture, the method comprising: a) providing a reaction cassettecomprising: (1) a reagent chamber having a first end and a second end,an exhaust port, a fluid port, and a retaining member, wherein a driedreagent is disposed in the reagent chamber at the second end, whereinthe exhaust port is at the first end and the fluid port is at the secondend, and wherein the retaining member is disposed in the reagent chamberbetween the exhaust port and the dried reagent and is sized such thatthe space between the interior of the chamber and the retaining memberis smaller than the dried reagent, obstructing passage of the driedreagent from the second end toward the first end of the chamber; and (2)a reaction chamber selectably in fluid communication with the reagentchamber, b) transferring a liquid through the fluid port into thereagent chamber to dissolve the dried reagent and form a solution; c)placing the reagent chamber in fluid communication with the reactionchamber; and d) transferring the solution from the reagent chamber intothe reaction chamber to assemble a reaction mixture.
 13. The method ofclaim 12, wherein the retaining member has a density less than 1,000kg/m³.
 14. The method of claim 12, wherein the dried reagent comprisesone or more lyophilized particulates.
 15. The method of claim 12,wherein the retaining member is fixed to the interior of the chamber.16. The method of claim 12, wherein the retaining member is movablydisposed in the chamber.
 17. The method of claim 16, wherein thelongitudinal axis of the chamber is in a vertical orientation with thesecond end being the bottom end and the first end being the top end, andwherein the retaining member is buoyant in the liquid.
 18. The method ofclaim 12, further comprising, between b) and c), moving the liquid inand out of the reagent chamber via the fluid port.
 19. A method fordissolving a dried reagent in a liquid, the reagent reservoircomprising: a) providing a reagent reservoir comprising a chamber, thechamber having (1) first and second ends, the second end being capableof holding the dried reagent; (2) an exhaust port at the first end; (3)a fluid port at the second end; and (4) a retaining member that isdisposed in the chamber between the first and second ends and is sizedsuch that space between the interior of the chamber and the retainingmember is smaller than the dried reagent, obstructing passage of thedried reagent from the second end toward the first end; and b)transferring the liquid through the fluid port into the second end ofthe chamber to dissolve the dried reagent in the liquid without thedried reagent passing the retaining member.
 20. The method of claim 19,wherein the retaining member has a density less than 1,000 kg/m³. 21.The method of claim 19, wherein the chamber further comprises a fixedmember immobilized in the chamber between the exhaust port and themovably disposed retaining member, the fixed member obstructing passageof the movably disposed retaining member through the chamber.
 22. Themethod of claim 19, wherein the dried reagent comprises one or morelyophilized particulates.
 23. The method of claim 19, wherein theretaining member is fixed to the interior of the chamber.
 24. The methodof claim 19, wherein the retaining member is movably disposed in thechamber.
 25. The method of claim 19, wherein the longitudinal axis ofthe chamber is in a vertical orientation with the first end being thetop end and the second end being the bottom end, and wherein theretaining member is buoyant in the liquid.
 26. The method of claim 19,further comprising, subsequent to b), moving the liquid in and out ofthe chamber via the fluid port.